Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, for producing methacrylic acid

Abstract

Provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway that can enhance the availability of reducing equivalents in the presence of methanol. Such reducing equivalents can be used to increase the product yield of organic compounds produced by the microbial organism, such as 3-hydroxyisobutyrate or MAA. Also provided herein are methods for using such an organism to produce 3-hydroxyisobutyrate or MAA.

What is claimed is: 
     
       1. A non-naturally occurring microbial organism comprising:
 (A) a methanol metabolic pathway, wherein said non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol or metabolize methanol as a carbon source for biosynthesis of methacrylate, wherein said methanol metabolic pathway comprises:
 (i) a methanol methyltransferase and a methylenetetrahydrofolate reductase; 
 (ii) a methanol dehydrogenase; or 
 (iii) a methanol dehydrogenase and a formaldehyde activating enzyme; and 
 
 (B) a methacrylate (MAA) pathway. 
 
     
     
       2. The non-naturally occurring microbial organism of  claim 1 , wherein said MAA pathway comprises at least one exogenous nucleic acid encoding one or more MAA enzymes expressed in a sufficient amount to produce MAA, wherein said MAA pathway comprises the MAA enzymes:
 (1) 2A, 2B, 2C, 2D, 2E and 2F; 
 (2) 2A, 2B, 2D, 2E and 2F; 
 (3) 2A, 2B, 2G and 2F; 
 wherein 2A is a succinyl-CoA transferase, ligase, or synthetase; 2B is a methylmalonyl-CoA mutase; 2C is a methylmalonyl-CoA epimerase; 2D is a methylmalonyl-CoA reductase (aldehyde forming); 2E is a methylmalonate semialdehyde reductase; 2F is a 3-hydroxyisobutyrate dehydratase; and 2G is a methylmalonyl-CoA reductase (alcohol forming). 
 
     
     
       3. The non-naturally occurring microbial organism of  claim 1 , wherein the non-naturally occurring microbial organism comprises two, three, four, five or six exogenous nucleic acids, each encoding a MAA pathway enzyme. 
     
     
       4. The non-naturally occurring microbial organism of  claim 3 , wherein said at least one exogenous nucleic acid encoding a MAA pathway enzyme is a heterologous nucleic acid. 
     
     
       5. The non-naturally occurring microbial organism of  claim 1 , wherein the methanol metabolic pathway comprises:
 (i) a methanol methyltransferase, a methylenetetrahydrofolate reductase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase, and a formyltetrahydrofolate deformylase: 
 (ii) a methanol methyltransferase, a methylenetetrahydrofolate reductase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase; 
 (iii) a methanol dehydrogenase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase; 
 (iv) a methanol dehydrogenase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase; 
 (v) a methanol dehydrogenase and a formaldehyde dehydrogenase; 
 (vi) a methanol dehydrogenase, an S-(hydroxymethyl)glutathione synthase, a glutathione-dependent formaldehyde dehydrogenase and an S-formylglutathione hydrolase; 
 (vii) a methanol dehydrogenase, a glutathione-dependent formaldehyde dehydrogenase and an S-formylglutathione hydrolase; 
 (viii) a methanol dehydrogenase, a formaldehyde activating enzyme, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase; or 
 (ix) a methanol dehydrogenase, a formaldehyde activating enzyme, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase. 
 
     
     
       6. The non-naturally occurring microbial organism of  claim 5 , wherein the methanol metabolic pathway further comprises a formate dehydrogenase, a formate hydrogen lyase, or a hydrogenase. 
     
     
       7. The non-naturally occurring microbial organism  claim 1 , wherein said non-naturally occurring microbial organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme. 
     
     
       8. The non-naturally occurring microbial organism of  claim 7 , wherein said at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme is a heterologous nucleic acid. 
     
     
       9. The non-naturally occurring microbial organism of  claim 1 , wherein the non-naturally occurring microbial organism further comprises one or more gene disruptions, said one or more gene disruptions occurring in one or more endogenous genes encoding protein(s) or enzyme(s) involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2 , and/or amino acids, by said non-naturally occurring microbial organism, and wherein said one or more gene disruptions confers increased production of MAA in said non-naturally occurring microbial organism. 
     
     
       10. The non-naturally occurring microbial organism of  claim 9 , wherein one or more endogenous enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2  and/or amino acids by said non-naturally occurring microbial organism, has attenuated enzyme activity or expression levels. 
     
     
       11. The non-naturally occurring microbial organism of  claim 1 , wherein the non-naturally occurring microbial organism further comprises a formaldehyde assimilation pathway, wherein said non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme expressed in a sufficient amount to produce an intermediate of glycolysis or a metabolic pathway that can be used in the formation of biomass, wherein said formaldehyde assimilation pathway comprises:
 (A) a hexulose-6-phosphate synthase and a 6-phospho-3-hexuloisomerase; or 
 (B) a dihydroxyacetone synthase and a dihydroxyacetone-phosphate kinase. 
 
     
     
       12. The non-naturally occurring microbial organism of  claim 11 , wherein the intermediate is:
 (A) a hexulose-6-phosphate, a fructose-6-phosphate, or a combination thereof; or 
 (B) a dihydroxyacetone, a dihydroxyacetone phosphate, or a combination thereof. 
 
     
     
       13. The non-naturally occurring microbial organism of  claim 11 , wherein the non-naturally occurring microbial organism comprises two exogenous nucleic acids, each encoding a formaldehyde assimilation pathway enzyme. 
     
     
       14. The non-naturally occurring microbial organism of  claim 11 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
       15. The non-naturally occurring microbial organism of  claim 1 , wherein said non-naturally occurring microbial organism is a species of bacteria, yeast, or fungus. 
     
     
       16. The non-naturally occurring microorganism of  claim 15 , wherein the non-naturally occurring microbial organism is selected from the group consisting of:
 (i)  Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens , and  Pseudomonas putida ; or 
 (ii)  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus , and  Rhizopus oryzae.    
 
     
     
       17. A method for producing methacrylate (MAA), the method comprising culturing the non-naturally occurring microbial organism of  claim 1  under conditions and for a sufficient period of time to produce methacrylate. 
     
     
       18. The method of  claim 17 , wherein the method further comprises separating the methacrylate from other components in the culture. 
     
     
       19. The method of  claim 18 , wherein the separation comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, or ultrafiltration. 
     
     
       20. The method of  claim 17 , wherein the non-naturally occurring microbial organism is cultured in a medium comprising biomass, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, starch, glycerol, methanol, carbon dioxide, formate, methane, or any combination thereof as a carbon source. 
     
     
       21. The method of  claim 17 , wherein the non-naturally occurring microbial organism is cultured in a medium comprising methanol as a carbon source. 
     
     
       22. The method of  claim 17 , wherein the non-naturally occurring microbial organism is cultured in a medium consisting essentially of methanol as a carbon source. 
     
     
       23. The method of  claim 17 , wherein the non-naturally occurring microbial organism is cultured in a substantially anaerobic culture medium.